The project has addressed the following areas in the past year: 1. Formation of a completely disordered protein complex. In collaboration with Ben Schuler (University of Zurich) and Birthe Kragelund (University of Copenhagen), we have been studying the formation of a complex between histone H1 and the protein prothymosin alpha, believed to act as a chaperone for H1. Experimentally, it was found that the complex has extremely high (picomolar) affinity, but with no evidence for formation of structure on binding. Using a minimalist coarse-grained model, we were able to reproduce the experimental FRET data accurately and to explain the NMR data. Our simulations show the key role of electrostatics in determining the structural ensemble of the bound complex. The complex is essentially completely disordered, with the propensities for contact formation correlating with local charge density in the sequence. The finding of such a high affinity complex which remains completely disordered represents a new paradigm in protein-protein interactions (Manuscript currently under revision in Nature). R. Best 2. Characterization of Alzheimer's Abeta monomer. The peptides Abeta40 and Abeta42 constitute the majority of the plaques found in the brains of Alzheimers patients. Understanding the mechanism of assembly of these plaques formed by fibrils of the Abeta peptides is therefore a major research objective. The starting point of assembly is of course the monomer; the finding of a very small primary nucleus for fibril formation (usually n=2) has suggested to some that conversion of the monomer to an aggregation-competent form may be a key step. Many studies, mostly from simulation, have suggested a compact, heterogeneous conformational ensemble in which stable secondary structures are formed, although there is some disagreement on the nature of the structures. We have collaborated with the group of Hoi-Sung Chung (LCP, NIDDK) to study structure formation in the monomer. FRET experiments in Chung's group showed a single state with no evidence for long lived stable species and a configurational relaxation time of 40 ns. All-atom simulations in our group also showed rapid configurational relaxation on a similar time scale and no evidence for populated collapsed or structured states. The simulations are in agreement with the measured FRET efficiencies as well as with hydrodynamic radii and NMR observables measured previously. Taken together, the data indicates the near complete absence of structured species in Abeta40 and Abeta42 monomers (manuscript in preparation). M. Bellaiche, R. Best 3. Folding of membrane proteins (Collaboration with Mark Sansom, University of Oxford). We are focussing our efforts on membrane protein folding using both coarse-grained and atomistic simulations, which is very challenging due to the high viscosity of lipid membranes. We find, using two-helix dimers as a testbed, that while current coarse-grained models yield reasonable values for dissociation constants, they often predict incorrect structures for the folded state (Ref 1). We have addressed the same problem with all atom simulations; in that case, the force field correctly predicts the structure of the native dimer, but with much too low stability. This was not previously appreciated due to the very extensive sampling needed for this (manuscript in preparation). J. Domanski, R. Best 4. Protein sequence evolution and design. Recent work has shown the potential of models parameterized on sequence alignments to capture correlations between residues in folded proteins. Such models intrinsically are able to assign a statistical energy to any sequence for its ability to fold into the structure corresponding to the sequence alignment. We have used such sequence-derived models in conjunction with statistical mechanics sampling methods to evaluate the total number of sequences which fold to a given structure (Ref 2). Taken to its logical extreme, this type of model could be used to design novel protein sequences, and we have tried this also, designing sequences which fold to each of the GA and GB domains of staphyloccocal protein G, as well as to an SH3 domain. We have now obtained well-folded examples of novel sequences folding to each of these domains by this approach, tested experimentally by John Louis (LCP, NIDDK), and we are aiming to determine NMR structures for representative examples of these proteins. In the final thread of the project, we are trying to design sequences which fold into different structures using the statistical models, thus formalizing an idea which has been pioneered empirically by Philip Bryan (University of Maryland). P. Tian, R. Best 5. Co-translational protein folding of titin. We are using coarse-grained simulations to interpret experiments in the group of Jane Clarke (Cambridge University) on the co-translational folding of titin on the ribosome. The experiments use an arrest peptide to stop translation by becoming stuck in the ribosome exit tunnel. However, there is a spontaneous rate of escape from the arrest, which may be increased by any force exerted by the protein. The yield of protein escaping the ribosome after a given incubation time is used as a measure of the force exerted. Using the forces measured in simulations, and experimental data on the force-dependence of the escape rate from earlier experiments, we have developed a kinetic model that allows us to compare directly with the yield of protein obtained in the experiment. The maximum yield is a trade-off between the maximum force exerted by the folded state (early in translation) and the population of the folded state (highest late in translation). P. Tian, R. Best 6. Interpretation folding psi-values. Psi-values have been proposed as an alternative to phi-values, in which a pair of histidine residues is engineered into a protein in order to create a divalent binding site for metal ions such as Zinc. However, several experimentalists have raised questions over the interpretation of psi-values. An additional concern is the time scale of ion association/dissociation relative to folding, since the time scale for ion binding is comparable to that of protein folding transition paths. We are using coarse-grained simulations to investigate under what experimental conditions psi values might be easily interpreted in terms of folding mechanism. W. Li 7. Formation of amyloid fibrils. We are working in collaboration with Tuomas Knowles (Cambridge and Sara Linse (Lund University) in order to describe the formation of amyloid fibers, and in particular the molecular mechanism of secondary nucleation. We are taking two-approaches. The first is to build a simple coarse-grained model based on the known structure of the fiber, which we are still working on. The second is to characterise the affinity, association rate and binding mode of the peptides with the surface of an existing fiber, which will be validated against mutant data from the Linse Lab. We have also obtained time on the Anton supercomputer in Pittsburgh to perform all-atom simulations of fibril elongation. W. Li, M. Bellaiche, R. Best 8. Development of coarse-grained models for protein phase separation (collaboration with Jeetain Mittal, Lehigh University). Recent work has shown that so-called stress granules formed in cells as a result of stress are a protein-rich phase which can be reconstituted in vitro from selected purified components. We have developed a coarse-grained model which can capture the sequence-dependence of the phase behaviour and the intermolecular interactions stabilizing the high density phase. Together with special simulation methods, we can determine phase diagrams from the model (Ref 3 & manuscript under review) W. Zheng, R. Best Group members involved in each project are listed at the end